Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.
Nat Methods
; 20(9): 1388-1399, 2023 09.
Article
in En
| MEDLINE
| ID: mdl-37474806
ABSTRACT
Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
CRISPR-Cas Systems
/
Gene Editing
Limits:
Humans
Language:
En
Journal:
Nat Methods
Journal subject:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Year:
2023
Document type:
Article
Affiliation country:
Germany